The association between high serum cholesterol levels and the incidence of cardiovascular disease has significantly increased the importance of understanding how the biosynthesis of cholesterol is regulated at a molecular level. It is known that cholesterol biosynthesis is regulated in vivo by regulation of the activity of the enzyme HMG-CoA reductase which catalyzes the "committed step" in cholesterol biosynthesis. Currently, experimental evidence has been presented which suggests regulation of the enzyme activity both by a regulation of enzyme concentration and possibly by a regulation of specific activity through phosphorylation. We have recently shown that the enzyme is extremely sensitive to inactivation by very low levels of disulfides. This observation coupled with the knowledge that in vivo levels of disulfides and thiols vary with the nutritional state, hormone levels, etc., suggests that HMG-CoA reductase activity may be modulated in vivo by a previously unconsidered mechanism of thiol/disulfide exchange. The long-range goal of the proposed research is to determine the importance of thiol/disulfide exchange to the regulation of HMG-CoA reductase activity in vivo. The specific experimental approach is to determine the chemical mechanism of disulfide inactivation of the enzyme, to measure the redox potential of the enzyme, and to develop an assay for determining the relative levels of oxidized (inactive) and reduced (active) HMG-CoA reductase. We will then determine if the measured levels of disulfides and thiols in rat liver under different metabolic conditions correlates directly with the extent of enzyme oxidation and enzyme activity. If regulation by this mechanism is observed, it may suggest several possibilities for drug design. If control by this mechanism is not observed, it would be important to determine why not since the enzyme is extremely sensitive to oxidiation by disulfides in vitro.